J. clin. Path., 1970, 23, 789-796 Effects of the venom of the rhinoceros horned J Clin Pathol: first published as 10.1136/jcp.23.9.789 on 1 December 1970. Downloaded from viper (Bitis nasicornis) on blood coagulation, platelet aggregation, and fibrinolysis N. MacKAY', J. C. FERGUSON, AND G. P. McNICOL From the University of East Africa, Medical School Extension, Kenyatta National Hospital, Nairobi, Kenya, the University Department of Materia Medica, Stobhill General Hospital, Glasgow, and the University Department of Medicine, The Royal Infirmary, Glasgow SYNOPSIS The venom of the rhinoceros horned viper (Bitis nasicornis) has been studied in vitro and has been shown to be anticoagulant. This action appeared to be due to an effect on both the extrinsic and intrinsic blood thromboplastin mechanisms. The venom was also proteolytic and in purified caseinolytic systems activated plasminogen, enhanced the activation of plasminogen by streptokinase, and potentiated the action of plasmin. In the euglobulin clot lysis system high concentrations of venom produced inhibition. The crude venom increased platelet adhesiveness but in high concentrations delayed the snowstorm effect in the Chandler's copyright. tube system and inhibited platelet iadenosine diphosphate reactivity. Passage through carboxy- methylcellulose yielded six fractions. One possessed anticoagulant activity, inhibited plasmin, and increased the optical density of platelet-rich plasma. The other five fractions shortened the plasma recalcification time but had no effect on plasmin activity. Four fractions aggregated platelets and enhanced platelet adenosine diphosphate reactivity. http://jcp.bmj.com/ The rhinoceros homed viper is distributed in a Methods band across Africa, stretching from West Africa to western Kenya (Bucherl, Buckley, and WHOLE BLOOD COAGULATION TIME Deulofeu, 1968). As its name implies it is charac- on September 28, 2021 by guest. Protected terized by a horn on its maxilla. The venom of This was determined as described by Biggs and this snake has been studied by Kirk and Corkill MacFarlane (1963). Ofthe venom solution, 0-1 ml (1946) who found that injected into mice the was added to each tube and in the control system venom produced local haemorrhage, and in the this was replaced by 0-1 ml saline. animals which died punctate haemorrhages were found in the kidneys and lungs. In vitro, the venom seemed to have no effect on the whole PLASMA RECALCIFICATION TIMES blood clotting time, but proteolytic activity was These were as described by Biggs and MacFarlane observed when the venom was incubated with (1963). In the test system various concentrations gelatin. of venom were substituted for saline. The present communication reports the findings of more detailed studies of the effects of the venom of the rhinoceros horned viper on the THROMBIN-FIBRINOGEN DILUTION CURVE haemostatic mechanism. The curve was constructed as described by Biggs and MacFarlane (1963). 'Please send requests for reprints to N. MacKay, Department of Materia Medica, Stobhill General Hospital, Glasgow, Wl. THROMBIN GENERATION TEST Received for publication 20 May 1970. The method of Pitney and Dacie (1953) was used. 790 N. MacKay, J. C. Ferguson, and G. P. McNicol J Clin Pathol: first published as 10.1136/jcp.23.9.789 on 1 December 1970. Downloaded from PROTHROMBIN CONSUMPTION TEST the test system. Adenosine diphosphate was Merskey's method (1950) was used. Saline or added to produce a final concentration of 0-5 venom in saline, 0 1 ml, was added. jug/ml. THROMBOPLASTIN GENERATION TEST PLATELET ADHESIVENESS To the standard test system of Biggs and Douglas This was measured by the method of Hellem (1953) was added 0-2 ml saline or 0-2 ml ofvenom (1960). solution in saline. The destruction of formed blood thromboplastin was measured by generating blood thromboplastin in the standard mixture VENOM FRACTIONATION for five minutes and adding 0 5 ml venom (or This procedure was carried out using a carboxy- saline). The decay of thromboplastic activity was methylcellulose column (CM 22). A total of then measured by transferring aliquots into sub- 150 mg venom in 3 75 ml of saline was applied to strate plasma at intervals. the top of the column and in a stepwise gradient barbital-acetate buffers of increasing pH (4 0-9 0) were used for elution. The effluent was constantly ONE-STAGE 'PROTHROMBIN TIMES' monitored in an ultraviolet spectrophotometer Venom (or saline), 0 1 ml, was added to the test (LKB Uvicord) and the protein-containing frac- system of Douglas (1962). tions were freeze dried after dialysis against distilled water for 24 hours at 4°C. EUGLOBULIN CLOT LYSIS TIME This was measured by the method of Nilsson and Olow (1962). Saline, 0 1 ml, was added to the Materials control system and 0.1 ml venom in saline in increasing concentration was added to the test system. The results are expressed in arbitrary Venom of Bitis nasicornis was supplied in dessi- units of activity derived from a double logarithmic cator dried form by Mr J. H. Leakey, Lake copyright. plot, 1 unit representing a lysis time of 300 Baringo, Kenya. Before use it was dissolved in minutes. saline. In the stock solution, venom was present in a concentration of 10 mg/ml. Purifiedpreparationsofhumanplasminogen and CASEINOLYTIC ASSAYS of plasmin, grade B, were supplied by A.B. Kabi, These were carried out by the method of Remmert Stockholm, Sweden, as also was streptokinase. and Cohen (1949) as modified by Alkjaersig Thrombin topical was supplied by Parke Davis, and adenosine diphosphate (ADP) by the Sigma (1960); the method is described by McNicol and http://jcp.bmj.com/ Douglas (1964), 0 1 M phosphate buffer being Chemical Company, St. Louis, Mo. substituted for acid and alkali. Where venom was used, 0 5 ml of a 10 mg/ml solution in saline was incorporated into the system in place of 0 5 ml of the phosphate buffer being used in the assay. Results Similarly, buffer was substituted for streptokinase and plasminogen in the experiments in which these WHOLE BLOOD COAGULATION TIME reagents were omitted. Results are expressed in Blood in a control tube to which saline had been on September 28, 2021 by guest. Protected casein units/ml. The effect on plasmin activity added coagulated in four minutes. The addition was assessed using a similar protocol. Plasminogen of venom in increasing concentrations caused and streptokinase were omitted and replaced by prolongation of the coagulation time and at a preformed human plasmin (A.B. Kabi, Stock- venom concentration of900 ,ug/ml the coagulation holm, Sweden). time was 11 minutes. PLATELET AGGREGATION IN THE PLASMA RECALCIFICATION TIMES CHANDLER'S TUBE The results are shown in Table I. At a final con- This was measured by a modification of the method of Chandler (1958) as described by Cunningham, McNicol, and Douglas (1965). Venom Concentration (;Lg/ml) Saline 3 13 52 208 PLATELET ADP REACTIVITY Clotting times (seconds) 116 115 180 325 >600 This was measured by the method of Born and Cross (1963), with the addition of 0-5 ml saline to Table I Plasma recalcification times with increasing the control system and 0 5 ml venom solution to concentrations of venom Effects of the venom of the rhinoceros horned viper J Clin Pathol: first published as 10.1136/jcp.23.9.789 on 1 December 1970. Downloaded from Fig. 1 Effect of various concentrations of venom on 16- rol thrombin generation. I/ 14- Fig. 2 Effect of venom on the interaction of thrombin andfibrinogen. 12- 133pgnL Fig. 3 Effect of various concentrations of venom on lo- I I \ \ thromboplastin generation. 8- }})/3n I *. ] 1/ \\\ centration of venom of 208 ug/ml the plasma 4. \\>~-wasW4 LqnkL rendered incoagulable. 2- THROMBIN GENERATION TEST The results are shown in Figure 1. In comparison 2 4 6 8 1o 12 14 6 with the control system thrombin generation was Fig. 1. rinm(minuts) progressively impaired and delayed by increasing the concentration of venom in the incubation 14 mixture. 12] A Salin THROMBIN-FIBRINOGEN REACTION IN THE 0 Venom PRESENCE OF VENOM In order to test the possibility that the venom was 10. ~~~~(2-5mg#/m) interfering with the conversion of fibrinogen to fibrin, human thrombin was generated fromcopyright. c 8- 1 4 1 I plasma and aliquots were transferred to two series c I I II of tubes, one containing fibrinogen-saline and the .| other fibrinogen-venom. The final concentration 0 of venom in the substrate was 2 5 mg/ml. The i' \ / \ \ results are illustrated in Figure 2. There is no evi- 4- dence that the venom interferes with the conver- sion of fibrinogen to fibrin under the influence of thrombin. http://jcp.bmj.com/ 2- PROTHROMBIN CONSUMPTION In the control system to which saline had been 2 4 6 8 10 added, the prothrombin consumption index was Fig. 2. Time(minutes) less than 5 %. When venom was present in a concentration of 900 ,ug/ml the index was 92%, on September 28, 2021 by guest. Protected indicating grossly defective prothrombin con- sumption. 70 60- SA" THROMBOPLASTIN GENERATION TEST The results are shown in Figure 3. With increasing s50 concentration of venom there was progressive c diminution in the amount of thromboplastin 40 - / / A X 1/^L detectable in aliquots from the incubation mixture. 30 - \ j 7./l/ g mi. DESTRUCTION OF FORMED BLOOD THROMBO- E 20 - PLASTIN 0 //3,uin.f,4 The results illustrated in Fig. 4 indicate that the 10- ,/ decay of formed thromboplastin is greater in the presence of venom than in the saline control. The 4M*5*.*LIs amount of thromboplastin detected is progress- >12 4 5 6* ively reduced as the concentration of venom in Fig. 3. rom.("inutm) the incubation mixture is increased. 792 N. MacKay, J. C.